The goal of the proposed research is to develop therapeutic reagents to block signaling by Sonic hedgehog (Shh). Aberrant Shh expression is implicated in pancreatic cancer, the 4th leading cause of cancer-related deaths in the US. In order to signal, Shh must be modified by attachment of the fatty acid palmitate to its N-terminus. We aim to exploit Shh palmitoylation as a potential Achilles heel by targeting Hhat (Hedgehog acyltransferase), the enzyme that catalyzes attachment of palmitate to Shh. Our laboratory used high throughput screening to identify RU-SKI 43, a novel, first-in-class small molecule Hhat inhibitor that blocks Shh palmitoylation, autocrine and paracrine Shh signaling, and human pancreatic cancer cell growth. We aim to further develop Hhat inhibitors into novel chemotherapeutics efficacious for the treatment of pancreatic and other cancers. This proposal also challenges the dogma that Shh action in pancreatic cancer is limited to paracrine signaling to the stroma. We have data that pancreatic tumor cells do indeed respond to Shh but via non-canonical, Smoothened-independent pathways. All of the Shh pathway inhibitors currently in clinical trials target Smoothened. The proposed R21 project has the potential to set a new direction in therapeutics by bringing the focus back to targets in the tumor epithelial cells. Aim 1. Optimization of Hhat inhibitors to regulate Shh palmitoylation and signaling The mechanism of action of Hhat inhibitors will be determined by monitoring signaling pathways regulated by Hhat and Shh in pancreatic cancer cells, as well as in estrogen-receptor positive breast cancer cells. Next, we will work on optimizing new, 2nd generation Hhat inhibitors that are up to 70x more potent than RU-SKI 43 in inhibiting Shh palmitoylation in vitro, but have short in vivo half-lives. Structure- activity relationships and a pharmacophore model developed by medicinal and synthetic chemists will be used for rational design and synthesis of 3rd generation and beyond Hhat inhibitors with increased potency and bioavailability. The efficacy of these inhibitors for blocking Shh palmitoylation, Shh signaling, and growth of human pancreatic cancer cells in vitro will be assessed. Aim 2. Hhat inhibitors as therapeutic agents to block Shh-driven cancers in vivo Experiments in this aim will be devoted to optimizing delivery of Hhat inhibitors into animals, by assessing pharmacokinetics and pharmacodynamics (ADME/PK/TOX) of the compounds. The ability of Hhat inhibitors to block tumorigenesis in a mouse xenograft model of pancreatic cancer, as well as in K- RasG12D/p53R172H mice, a genetically engineered mouse model of pancreatic cancer, will be assessed.